delta12-13, 17-seco-androstene compounds and lactone intermediates therefor



United States Patent This invention relates to the synthesis of new steroids and derivatives thereof, and more particularly to the preparation of new testololactones and seco-androstatriencic acids.

The final compounds of this invention are of the general formula -CH CH COOR wherein X is hydrogen, chloro or fluoro (preferably hydrogen or fiuoro), and R is hydrogen or a hydrocarbon radical of less than ten carbon atoms, as exemplified by lower alkyl (e.g., methyl, ethyl, propyl, butyl, hexyl and octyl), monocyclic ar(lower alky-l) (e.g., benzyl, phenethyl, 3-phenylpropyl, 4-phenylbutyl, and o, In, or p-tolylethyl), monocyclic aryl, cycloalkyl, cycloalkenyl, cycloalkyl(lower alky), lower alkenyl, and monocyclic ar- (lower alkenyl).

The final compounds of this invention, wherein R is hydrogen, can be obtained directly by culturing Cylindr carpon radicicola in the presence of either ll-ketoprogesterone or a 9a-halo-ll-ketoprogesterone, whereby a mixture of the desired final product and either ll-keto-l-dehydrotestololactone or 9oc-flll0l0 (or chloro)-ll-keto-ldehydrotestololaetone is produced; or indirectly from said 1-dehydrotestololactones by treatment of the latter with a base.

The method of culturing Cylindrocarpon radicicola for the purpose of this invention is the same as that described in US. Patent No. 2,868,694, except that either ll-keto progesterone or a 9or-halo-1l-ketoprogesterone is employed as the steroid substrate. That is, the steroid is added to a growing culture of Cylindrocarpon radicicola either during the incubation period or by including it in the nutrient medium prior to inoculation. if 11-keto progesterone, 9ot-iodo-ll-ketoprogesterone, or 9abromoll-ketoprogesterone is employed as the substrate, M 13,l7-seco androstatriene-3,11-dione-l7-oic acid is obtained as the product. If, however, 9a-fluoro-lldcetoprogesterone is used as the substrate, 9afl.lO1O-A -13,- 17-seco-androstatriene-3,11-dione-l7-oic acid is obtained; and 9a-ch10r0-1 l-ketoprogesterone yields 9a-ohloro-A 13,17-seco-androstatriene-3,11-dione-l7-oic acid.

These free acids can then be converted to their ester derivatives in the usual manner, as by treatment with a desired alcohol in the presence of an esterification catalyst or, if a lower alkyl ester is desired, by treatment with a diazo (lower alkane), such as diazomethane.

The final compounds of this invention are physiologically active substances which possess protein-anabolic activity, and hence can be used in lieu of known proteinanabolic steroids, and may be administered either perorally or parenterally in the treatment of post-operative shock and other conditions where tissue degeneration has occurred, being formulated for such administration in the same type of preparations as testosterone, for example, with concentration and/or dosage based on the activity of the particular compound.

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As stated hereinbefore, in addition to the seco-androstatrienoic acids, the process of this invention employing Cylindrocarpon radicicola as the microorganism yields 1 l-ketol-dehydrotestololactone, 9a-cl1l0r0-1l-keto-l-dehydrotestololactone, or 9a-fiuoro-1l-keto-l-dehydrotestololactone, depending on whether the steroid substrate is ll-lcetoprogesterone (or 9a-iodo-1l-ketoprogesterone or 9a-bromo-1 l-ketoprogesterone) 9a-chlor0-l l-ketoprogesterone, or 9oc-iuoro-1l-ketoprogesterone, respectively.

These 1l-keto-l-dehydrotestololactones are new compounds of this invention which may be converted to the final products of this invention by treatment with a strong base, such as an alkali metal hydroxide (e.g., potassium hydroxide and sodium hydroxide). The reaction is preferably conducted at an elevated temperature.

The 11-l eto-l-dehydrotestololactone intermediates of this invention can also be prepared by a two-step process of this invention. In the first step, ll-ketoprogesterone, 9a-chloro-1l-ketoprogesterone or 9u-fluoro-11-ketoprogesterone is subjected to the action of Penicillium citrinum, whereby ll-ketotestololactone, 9a-chloro-11-ketotestololactone, and 9zx-fluoro-ll-ketotestololactone are formed, respectively. These ll-ketotestololactones are new intermediates of this invention.

The conversion is effected by either bringing together, in an aqueous medium, the steroid, oxygen, and enzymes of non-proliferating cells of Penicilliam citrinum or, preferably, by including the steroid in an aerated culture of Penicillium citrinum.

W'hen aerated culture is used, the oxidation is effected in the presence of Penicillium citrinum by adding the steroid to the culture during the incubation period, or by including it in the nutrient medium prior to inoculation. In any case, assimilable sources of nitrogenous materials for growth promotion and carbon-containing substances as energy sources should be present in the culture medium. Also, an adequate air supply should be maintained during the oxidation, e.g., by the conventional techniques of (l) exposing a large surface of the medium to air or (2) aerating in submerged culture.

In. general, the conditions of culturing Penicillium citrz'num for the purpose of this invention are (except for the inclusion of the steroid) the same as those of culturing such microorganism for the production of other metabolites. Thus, the nutrient medium essentially comprises assirnilable sources of nitrogen for growth and carbon for energy.

The nitrogen source materials may be organic (e.g., soybean meal, cornsteep liquor, meat extract, and/ or distillers solubles) or synthetic (i.e., composed of simple, synthesizable organic or inorganic compounds such as ammonium salts, alkali nitrates, amino acids, urea or thiourea).

As to the energy source material, lipids, especially (1) fat acids having at least 14 carbon atoms, (2) fats or (3) mixtures thereof, are preferred. Examples of such fats are lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein and triiaurin; and illustrative fat acids include stearic, palmitic, oleic, linoleic and myristic acids.

Other carbon-containing materials may also be used. For example such materials as glycerol, glucose, fructose, sucrose, lactose, maltose, dextrins, starches, whey, etc., are adequate carbon source materials. These materials may be used either in purified state or as concentrates, such as whey concentrate, corn, wheat or barley mash; or mixtures of the above may be employed. It is to be noted, however, that the steroid is added to the fermentation medium essentially as a precursor and not as an energy source.

The ll-ketotestololactones thus formed can then be dehydrogenated in the 1,2-position, either microbiologically, as by treatment with a known l-dehydrogenating microorganism such as N ocardia restrictus, or chemically, as by treatment with selenium dioxide, or 2,3-dichloro- 5,6-dicyanobenzoquinone to yield the ll-keto-l-dehydrotestololactones of this invention.

In addition to their use as intermediates, the ll-ketotestololactones of this invention, that is compounds of the general formula ample, with concentration and/or dosage based on the activity of the particular compound.

The following examples illustrate the invention (all temperatures being in centigrade):

EXAMPLE 1 Surface growth from a three-week old agar slant culture of Cylindrocarbon radicicola (ATCC 11011), the slant containing as a nutrient medium (A): glucose, g.; Difco yeast extract, 2.5 g. K HPO 1 g.; agar, g.; and distilled water to 1 1., is suspended in 2.5 ml. of a 0.01% sodium lauryl sulfate aqueous solution. One milliliter portions of the suspension are used to inoculate two 250 ml. conical flasks, each containing 50 ml. of the following sterilized nutrient medium (B): dextrose, 10 g.; cornsteep liquor, 6 g.; NH H P0 3 g.; Difco yeast extract, 2.5 g.; CaCl 2.5 g.; and distilled water to 1 1. After 48 hours of incubation at with continuous rotary agitation (280 cycles per minute, 2 inch radius), 10% (vol./vol.) transfers are made to twelve 250 ml. conical flasks each containing 50 ml. of fresh sterilized medium B. These are incubated under the conditions described above for 24 hours, after which another 10% (vol/vol.) transfer is made to 100 additional 250 ml. flasks containing 50 ml. of fresh sterilized medium B. The 9a-fluoro-11-ketoprogesterone is added by adding to each flask 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide (60 mg./ml.) so that the medium is supplemented with 300 ,ug./ml. of steroid. After 48 hours of further incubation, the contents of the flasks are pooled and filtered through a Seitz clarifying pad. The flasks, myceliurn and pad are washed With successive 50 ml. portions of Warm water. The combined filtrate and washings (pH 8.2) are acidified to pH 4 with acetic acid and extracted three times with 2 1. portions of chloroform. The combined chloroform extracts are then washed twice with 3 l. portions of water and evaporated to dryness, in vacuo. The residue is redissolved in 200 ml. of ethyl acetate and extracted with 2 x 100 ml. portions of 5% sodium bicarbonate. The ethyl acetate is then washed with water, dried over sodium sulfate and evaporated to dryness, in vacuo. Crystallizations of the residue from acetonehexane gives about 100 mg. of 9oc-fiuoro-1l-keto-n -dehydrotestololactone having a melting point of about 217-220"; [a] +28.5 (chlf);

us 23 1 me (e =16,000); N525? mm 4 u 27,000); mg? 5.80, 5.86, 6.00, 6.13, 622,11; E32 1.19 volts (vs. mercury pool anode) Analysis.Calcd. for C H O F (332.48): C, 68.66; H, 6.37. Found: C, 68.95; H, 6.12.

The sodium bicarbonate extracts are acidified with 2 N hydrochloric acid and extracted with three ml. per tions of chloroform. The combined chloroform extracts are then washed with water and evaporated to dryness, in vacuo. Crystallization of the residue from acetone hexane gives about 910 mg. of 9cz-fluoro-A f -13,17 seco androstatriene-3,11-dione-17-oic acid having a melting point of about 217220; [M 46.5 (0111f); rag 210 m ($28,500); mi 5.80, 6.02, 6.12, 6.24 E55 1.14 volts (vs. mercury pool anode) Analysis.Calcd. for C H O F (332.48): C, 68.66; H, 6.37. Found: C, 69.03; H, 6.21; neut. eq. 347

EXAMPLE 2 Following the procedure of Example 1 but substituting an equivalent amount of 9a-chloro-1l-ketoprogesterone for the 9d-fluoro-1l-ketoprogesterone, 9a-chloro-l1-keto l-dehydrotestololactone and 9a-chloro-A -13,17-secoandrostatriene-3,1l-dione-17-oic acid are obtained.

EXAMPLE 4 11-ket0-1-dehydrotesrololactone and 11 43,] 7-secoandrostatriene-SJ1-di0ne-17-0ic acid Following the fermentation and extraction procedure described in Example 1 but substituting ll-ketoprogesterone for the 9ot-fluoro-ll-ketoprogesterone, there is obtained about 320 mg. of 11-keto-A -dehydrotestololactone having MP. about 196-198"; M1 +79.0 (chlf);

M13}, 238 ma (6 =15,900) k952i? W) 239 mu (e 25,600); x 5.74, 5.83, 6.00, 6.13, 6.22,.

Analysis.-Calcd. for C H O (314.37): C, 72.59; H, 7.05. Found: C, 72.51; H, 7.24; and about 363 mg. of A -13,17-seco androstatriene-3,11-dione-17-oic acid having MP. about 167-169; [M i0 (chlf.);

x513; 258 m1. (e=29,800); 5.81, 6.00, 6.02 (sh.), 6.22 811. 6.24,.

Nuiol max.

EXAMPLE 5 The fermentation is carried out as described in Example 1 except that 9a-bromo-11-ketoprogesterone is used as the substrate and the final stage of the fermentation is carried out in 2 l. flasks containing 500 ml. of medium. The products are separated into neutral and acidic fractions as described in Example 1 to give upon crystallization about 500 mg. of l1-keto-A -dehydrotestololactone and about 200 mg. of A -13,l7-seco-androstatriene-3,11-di0ne-17- oic acid identical with the products of Example 4.

Similarly, 9oc-iOdO-1 l-ketoprogesterone yields ll-ketod -dehydrotestololactone and A -13,l7-seco-andro statriene-3,1l-dione-17-oic acid.

in l.

EXAIVIPLE 6 60 mg. of 9u-fluoro-11-keto-A -dehydrotestololactone is dissolved in 5 ml. of 5% potassium hydroxide in methanol and the solution is warmed on a steam bath for five minutes. It is then cooled, neutralized with acetic acid, diluted with water and extracted with ether. The ether is then re-extracted with 5% sodium bicarbonate which is acidified and extracted with ether. The ether is dried and evaporated to dryness and the residue crystallized from acetone-hexane to give 9a-fluoro-A -13,17-secoandrostatriene-3,11-dione-l7-oic acid.

Analysis.Calcd. for C H O F (332.48): C, 68.64; 6.37; F, 5.71. Found: C, 67.78; H, 6.81; F, 5.67.

EXAMPLE 7 A -J3J7-sec0-andr0statriene-3,11-di0ne-17-0ci acid Following the procedure of Example 6, but substituting an equivalent amount of 1l-keto-A -dehydrotestololac tone for the 9oc-fiuoro-1l-keto-A -dehydrotestololactone, A -13,l7-seco-androstatriene-3,1l-dione-l7-oic acid is obtained.

EXAMPLE 8 To a suspension of 360 mg. of 9OLfll.lOI'O-A -13,17- seco-androstatriene-3,l1-dione-17-oic acid in 10 ml. of methanol, an ethereal solution of diazomethane is added slowly with swirling until the steroid dissolved and the yellow color of the diazomethane persists. After 30 minutes at room temperature the solution is evaporated, in vacuo, and the residue distributed between ethyl acetate and 5% sodium bicarbonate. The ethyl acetate is washed well with water, dried over sodium sulfate and evaporated to dryness, in vacuo. Chromatography of the residue on Woelm neutral alumina and elution with chloroformbenzene (1:9) gives on crystallization from acetonehexane about 200 mg. of methyl 9oz-fltlOIO-A -l3,17- seco-androstatriene-3,11-dione-17-oate, melting point about 1l9121; -46.9 (chlf.);

Analysis.Calcd. for C H O F (346.38): C, 69.34; H, 6.69; F, 5.49. Found: C, 69.50; H, 7.09; F, 5.53.

EXAMPLE 9 Methyl A -13,17-sec0-andr0statriene-3,11-

dione-l 7-0ate Following the procedure of Example 8, but substituting an equivalent amount of ti 43,17-seco-androstatriene- 3,11-dione-17-oic acid for the 9a-fiuoro steroid, methyl A -13,l7-seco-androstatriene-3,11-dione-l7-oate is obtained.

XAMPLE 10 Following the fermentation and extraction procedures described in Example 1 but substituting a culture of Penicillium citrinum (ATCC 8506) for the C. radicicola there is obtained on crystallization of the neutral fraction about 667 mg. of 9ct-fluoro-1l-ketotestololactone, M.P. about 193-195"; [ed +59.4 (chlf.); A233,, 233 my (6 =17,400); Ami? 5.77, 6.00, 6.17 1.

Similarly, 9a-chloro-ll-ketoprogesterone yields 90(- chloro-l l-ketotestololactone.

EXAMPLE 11 11 -ket0testol0lact0ne Following the fermentation and extraction procedures described in Example 1 but substituting Penicillium 6 citrinum (ATCC 8506) for C. radicicola and ll-ketoprogesterone for 9a-fiuoro-ll-ketoprogesterone there is obtained about 650 mg. of ll-ketotestololactone having a melting point of about 226-228, +121 (chlf.);

A315, 237 In,u (e =17,200) M223, M603 238 my (6 27,400); was? 5.81, 5.84., 6.00, 6.17

Analysis.Calcd. for C H O (316.38): C, 72.12; H, 7.65. Found: C, 72.26; H, 7.77.

EXAMPLE 12 9a-flu0r0-11-keto-A -dehydr0testololactone The fermentation is carried out as described in Example 1, however, a culture of Nocardia restrictus (Culture Collections, Rutgers Institute of Microbiology, No. 545) is substituted for the C. radicz'cola and 200 mg. of 9mfluoro-ll-ketotestololactone is used as the substrate and distributed in twenty 250 m1. flasks. The fermentation is allowed to proceed for 7 /2 hours, then filtered and washed with water and the combined filtrate and washing 1.1 l.) extracted three times with 300 ml. portions of chloroform. The chloroform extracts are combined, washed with water and evaporated to dryness, in vacuo. Crystallization of the residue from acetone-hexane gives about 100 mg. of 9ix-fluoro-1l-keto-A -dehydrotestololactone.

Similarly, 91x-chloro-1l-ketotestololactone yields chloro-l 1-keto-A -dehydrotestololactone.

EXAMPLE 13 1 1 -ket0-A -dehydrotestololactone CH OH C 0 OR wherein R is selected from the group consisting of hydrogen and an unsubstituted hydrocarbon radical of less than ten carbon atoms.

8. A compound of the formula wherein R is selected from the group consisting of hydrogen and an unsubstituted hydrocarbon radical of less than ten carbon atoms.

(References on following page) References Cited by the Examiner UNITED STATES PATENTS 8 FOREIGN PATENTS 792,803 4/58 Great Britain.

Picha 260343-2 OTHER REFERENCES Knowles 260-3432 p Picha 5 Fleser t aL. hrmds, Rumhold Publ. C0,, New York (1959), page 475.

Npbfle 260*3432 Fieser et 211.: Steroids, Reinheld Pub}. Corp, New Blble 26514-5 Y rk 1959 pages 592 and 593.

Mprray et 195 51 Herr et a1.: Jour. Amer. Chem. 800., vol. 78, pages 500 Fned et a1. 260.343.2 10 and 501 Thoma et a1. 19551 Wendler 260514.5

IRVING MARCUS, Primary Examiner.

NICHOLAS S. RIZZO, Examiner. 

6. 9A-CHLORO-11-KETO-TESTOLOLACTONE.
 7. A COMPOUND OF THE FORMULA 